依赖利福平结核分枝杆菌相关蛋白的氨基酸及其编码基因序列研究

目的 研究临床依赖利福平结核分枝杆菌（依R菌）相关蛋白的氨基酸及其编码基因序列。方法以罗氏绝对浓度法药敏实验鉴定临床依R菌株（49株）、耐R菌株（54株）和R敏感菌株（30株），用十二烷基硫酸钠-聚丙烯酰胺电泳（SDS-PAGE）法对不同临床菌株及H37Rv标准株共计134株菌株的菌体蛋白质图谱进行比较分析，通过Q-TOF2型毛细管液相色谱-电喷雾-四极杆-飞行时间串联质谱仪（LC-ESI-MS-MS）对其差异表达蛋白的氨基酸序列进行鉴定，并用对其蛋白的编码基因进行DNA序列分析。结果不同菌株的SDS-PAGE菌体蛋白图谱基本相似。49株依R菌有4l株（84％）在含R管中的菌体蛋白图谱有一条相同的高表达蛋白带（相对于标准物分子质量43000的下端），约占总菌体蛋白的30％～50％，H37Rv标准株、28株R敏感菌（28／30）及44株耐R菌（44／54）的含R管和对照管的蛋白图谱相似，且无相应的高表达现象；该高表达蛋白经氨基酸序列鉴定为结核分枝杆菌H37Rv假设蛋白Rv0341，相对分子质量为43894，等电点（PI）5．25，得分183。不同菌株的Rv0341编码基因1440bp扩增片段，经DNA测序分析均未发现突变。结论利福平对Rv0341在依R菌中的表达有上调作用，Rv0341与依R菌相关，可视为依R菌的相关蛋白或标志物；Rv0341与依R菌的毒力及细胞壁的合成是否相关有进一步的研究价值。     

结核分枝杆菌毒力分泌系统Rv3871基因的分子克隆及蛋白表达研究

目的为探索参与结核分枝杆菌(MTB)毒力分泌系统相关基因的分子功能,对MTB H37Rv株编码Rv3871蛋白基因进行克隆、表达及鉴定分析。方法以结核分枝杆菌H37Rv基因组为模板,用PCR扩增Rv3871基因片段,并重组到原核表达载体pET32a( )中,转化E.coliBL21(DE3)菌株,用IPTG诱导蛋白表达,通过SDS-PAGE和Western blot对目的蛋白进行检测分析。结果阳性重组质粒经BamHⅠ和XhoⅠ双酶切得到1 776和5 900 bp两条片段,与预期大小一致。重组质粒测序,与NCBI上Rv3871编码序列比对完全一致,且输入片段读码框与表达载体读码框相吻合。SDS-PAGE检测表达蛋白分子质量单位为84 ku;Western blot检测出特异性阳性信号。结论本研究成功构建了结核杆菌毒力分泌系统重要相关基因Rv3871的原核表达质粒,并在大肠埃希菌中表达Rv3871蛋白,为进一步探讨该基因在MTB毒力分泌中的功能奠定了基础。     

对利福平耐药的结核分枝杆菌rpoB基因突变与耐药程度关系的探讨

目的　探讨对利福平耐药结核分枝杆菌的rpoB基因的突变位置与耐药程度的关系。方法　 32株对5 0 μg·mL-1的利福平耐药 (R5 0 )、2 2株对 2 5 0 μg·mL-1的利福平耐药 (R2 5 0 )、10株对利福平、异烟肼、乙胺丁醇及链霉素敏感的结核分枝杆菌株 (R0 )和 1株H3 7Rv结核分枝杆菌株包括核心区域在内的rpoB基因片段进行了DNA序列分析。结果　 (1) 10株 (R0 )对利福平、异烟肼、乙胺丁醇及链霉素敏感的结核分枝杆菌株和 1株H3 7Rv结核分枝杆菌株未检测到rpoB基因易突变区的改变 ;(2 )耐利福平的低耐药 (R5 0 )结核分枝杆菌突变 2 5株 (78 1% ) ;耐利福平的高耐药 (R2 5 0 )结核分枝杆菌突变 2 1株 (95 5 % ) ,两者差异无显著性 (P =0 170 ) ;(3)耐利福平的低耐药 (R5 0 )结核分枝杆菌突变点呈散在分布 ;(4 )耐利福平的高耐药 (R2 5 0 )结核分枝杆菌突变以 5 31位氨基酸突变为主 (5 7 1% )以及联合突变较多见 (2 3 8% ) ,与R5 0相比两者差异有显著性。结论　大部分对利福平耐药的结核分枝杆菌均有rpoB基因突变。在对利福平低耐药结核分枝杆菌的rpoB基因中 ,其突变位置呈散在分布。在对利福平高耐药结核分枝杆菌的rpoB基因中 ,5 31位密码子突变及多个密码子联合突变是其主要突变特征。     

结核分枝杆菌Rv1884基因的克隆、表达及亲和层析纯化

目的　克隆表达结核分枝杆菌Rv1884基因 ,序列测定正确后进行融合表达、纯化。方法　采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv基因组中扩增出Rv1884编码基因 ,用限制性内切酶消化后插入到pGEM Teasy中 ,序列测定正确后 ,再亚克隆到融合表达载体 pPro EXHT中 ,转化大肠杆菌DH5α ,目的基因经IPTG诱导 ,由T7启动子调控表达了氨基端带 6个连续组氨酸残基的Rv1884蛋白 ,在变性条件下对目的蛋白进行纯化。结果　获得了结核分枝杆菌H37Rv株Rv1884蛋白基因 ,得到融合 6个组氨酸残基的Rv1884蛋白纯度大于 85 %。结论　构建了结核分枝杆菌Rv1884基因的重组表达载体 ,并获得了高纯度的融合表达蛋白 ,为以后的深入研究奠定了基础。     

结核分枝杆菌异烟肼耐药基因katG的检测

目的探讨结核分枝杆菌异烟肼(INH)耐药的分子机制,建立快速检测结核分枝杆菌INH耐药性的方法。方法应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测105株结核分枝杆菌临床分离株的katG基因,运用DNA测序和生物信息分析比对进行验证。结果以H37Rv标准株为对照,34株敏感株中5株(14.7%)存在KatG基因异常,105株耐药株中55株(52.4%)存在KatG基因异常。结论结核分枝杆菌对INH耐药与KatG基因突变有关,PCR-RFLP技术可快速、准确地检测结核分枝杆菌对INH的耐药性。     

结核分枝杆菌分泌蛋白MPT53基因的克隆、表达和纯化

目的构建结核杆菌分泌蛋白MPT53原核表达载体并进行表达和纯化。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板扩增MPT53基因，产物经纯化回收后与载体pMD-T连接转化，酶切鉴定，克隆到pET32a原核表达载体，测序鉴定插入序列完全正确者转化大肠埃希菌BL21，诱导表达MPT53融合蛋白，亲和层析纯化后，进行SDS-PAGE电泳鉴定。结果成功构建PET32a-MPT53重组质粒，转化BL21后经IPGT诱导成功表达分子质量单位为36ku的MPT53蛋白，与理论值相符。结论成功表达结核分枝杆菌MPT53蛋白，为其应用打下了基础。     

结核分枝杆菌Rv0577基因在毕赤酵母的表达及重组抗原活性鉴定

目的在毕赤酵母表达结核分枝杆菌Rv0577基因并鉴定重组蛋白抗原活性。方法从结核分枝杆菌标准株H37Rv扩增Rv0577基因,TA克隆进入pGEM-TEasy载体,再亚克隆进入pPICZαA。重组质粒pPICZαA-Rv0577经双酶切鉴定并测序证实,线性化后电转化导入毕赤酵母X-33表达;采用Western blot和Dot blot鉴定抗原活性。结果双酶切及测序鉴定均证实获得Rv0577基因的正确克隆。pPICZαA-Rv0577在X-33分泌表达的目的蛋白,通过硫酸铵沉淀、HPLC顺序纯化,获得的纯化蛋白与结核病患者血清进行Dot blot,7份中有3份反应阳性。结论Rv0577基因在毕赤酵母获得高效表达,重组抗原可被结核病患者血清识别,具有抗原活性。     

结核分枝杆菌Rv1009基因蛋白的克隆和表达

目的克隆表达结核分枝杆菌促Rv1009基因,序列测定正确后进行融合、表达。方法采用热启动聚合酶链反应(Polymerase Chain Reaction,PCR)从结核分枝杆菌H37Rv基因组中扩增出Rv1009编码基因,用限制性内切酶消化后插入pGEX 4T-2载体中,将重组质粒转化大肠杆菌BL21 (DE3),目的基因经IPTG诱导,表达Rv1009基因蛋白。结果经PCR扩增在1300bp处发现一条目的片段,获得了结核分枝杆菌H37Rv株Rv1009基因蛋白,经诱导后高效表达分子量为64KD的外源蛋白,与预期分子量大小一致,凝胶自动扫描分析,在A600值为0．6,IPTG终浓度为0．3 mmol／L,诱导表达3 h时融合蛋白表达量即达峰值,占菌体总蛋白的22．8％。结论构建了结核分枝杆菌Rv1009基因重组表达载体,获得了RPF样融合蛋白的高效表达,为今后深入研究奠定了基础。     

结核分枝杆菌分泌蛋白MPT64的克隆表达和纯化

目的构建结核分枝杆菌分泌蛋白MPT64原核表达载体并进行表达和纯化。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板扩增出MPT64基因,产物经纯化回收后与载体pMD-T连接转化,酶切鉴定,克隆到pET21a原核表达载体,测序鉴定插入序列完全正确者转化大肠杆菌BL21,诱导表达MPT64融合蛋白,利用亲和层析纯化表达产物,SDS-PAGE电泳进行鉴定。结果成功构建MPT64表达载体,SDS-PAGE电泳鉴定成功表达MPT64蛋白。并以此蛋白进行结核抗体的检测,其特异性和敏感性分别为96%和43%。结论成功表达结核分枝杆菌MPT64蛋白,并进行特异性和敏感性的检测,证明重组的MPT64蛋白有希望成为结核病血清学诊断的组合抗原的候选者之一。     

结核分枝杆菌Ag85B+Rv3407融合基因自杀性DNA疫苗的构建及鉴定

目的构建表达结核分枝杆菌生长期抗原Ag85B和休眠期蛋白Rv3407的自杀性DNA疫苗融合基因表达载体,并检测其在BHK-21细胞中的表达。方法采用PCR方法从结核分枝杆菌基因组中扩增Ag85B基因和Rv3407蛋白基因,再以二者的混合物为模板扩增Ag85B+Rv3407融合基因,构建真核表达载体pSCA1/Ag85B+Rv3407,以ELISA法检测其在BHK-21细胞中的瞬时表达。结果从结核分枝杆菌基因组中扩增得到Ag85B+Rv3407融合基因,片段大小为1 323bp,与预期相符。成功构建重组质粒pSCA1/Ag85B+Rv3407,该重组质粒能在BHK-21细胞中表达目的蛋白(实验组P/N≥2.1)。结论成功构建结核分枝杆菌自杀性DNA疫苗融合基因表达载体,该重新载体能在真核细胞中表达目的蛋白。     

结核分枝杆菌链霉素耐药基因rpsL的检测

目的探讨结核分枝杆菌(TB)对链霉素(Sm)耐药的分子机制,建立快速检测结核分枝杆菌Sm耐药性的方法。方法应用聚合酶链反应-单链构象多态性(PCR-SSCP)和聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测80株结核分枝杆菌临床分离株的rpsL基因。结果以H37Rv标准株为对照,21株敏感株中,1株(4.76%) SSCP和RFLP分析存在rpsL基因异常;59株耐药株中,36株(61.02%)SSCP和RFLP分析存在rpsL基因异常。结论结核分枝杆菌对Sm耐药与rpsL基因突变有关,PCR-SSCP和PCR-RFLP技术可快速、准确地检测结核分枝杆菌对Sm的耐药性。     

Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c.

SETTING: The PPE gene family of Mycobacterium tuberculosis is thought to be of immunological significance. One member, Rv1917c, is highly polymorphic in clinical isolates. OBJECTIVE: To characterize Rv1917c gene polymorphism and expression, and to determine the cellular location and glycosylation status of the encoded protein. DESIGN: Tandem repeat regions of Rv1917c were amplified and sequenced to determine the molecular basis for the gene polymorphism. RT-PCR analysis was utilized to detect expression of Rv1917c mRNA in liquid cultures of M. tuberculosis H37Rv. The gene was cloned as a 3'-terminal green fluorescent protein (GFP) fusion, downstream of an acetamide-inducible promoter, and expressed in Mycobacterium smegmatis and Mycobacterium bovis BCG. The expression product was characterized in terms of cellular location and glycosylation status. RESULTS: PCR and sequence data demonstrated that variable numbers of tandem repeats within Rv1917c contribute to gene polymorphism. RT-PCR analysis demonstrated that Rv1917c mRNA is expressed in liquid cultures of M. tuberculosis H37Rv. Expression of the recombinant protein in M. smegmatis and M. bovis BCG was visualized by fluorescence microscopy and flow cytometry. A protein of the predicted size (166 kDa) was confirmed by Western blotting. Cell fractionation studies demonstrated that the recombinant protein is hydrophobic, suggestive of cell wall-association, while flow cytometric data derived from antibody binding experiments suggested that it is surface exposed. Analysis of the glycosylation status of the expressed protein failed to demonstrate glycosylation. CONCLUSION: Rv1917c mRNA is expressed in M. tuberculosis H37Rv, and Rv1917c gene polymorphism is associated with variable numbers of tandem repeats. The recombinant Rv1917c protein is surface exposed.     

Inactivation of the Mycobacterium bovis homologue of the polymorphic RD1 gene Rv3879c (Mb3909c) does not affect virulence

The RD1 locus is deleted from all strains of Mycobacterium bovis BCG but present in virulent isolates of M. bovis and Mycobacterium tuberculosis. The RD1 gene Rv3879c encodes a proline- and alanine-rich protein that shows sequence polymorphism across members of the M. tuberculosis complex. The role of this protein in virulence was investigated by deleting the Rv3879c homologue from M. bovis (Mb3909c) and testing the virulence of the mutant in the guinea pig model. The M. bovis Delta Mb3909c mutant was not attenuated in the guinea pig model, showing that this gene does not encode a virulence factor and plays no role in the attenuation caused by loss of RD1.     

应用基因阵列法快速检测结核分枝杆菌rpoB基因突变

目的　研制一种新型的基因阵列 ,用于结核分枝杆菌耐利福平分离株rpoB基因突变的快速检测。方法　根据结核分枝杆菌rpoB基因序列设计寡核苷酸探针并制作基因阵列 ,用生物素标记的引物扩增结核分枝杆菌rpoB基因突变热点的目的片断 ,与基因阵列杂交 ,同时以聚合酶链反应 单链构象多态性 (PCR SSCP)技术及DNA测序法为对照。结果　 111株结核分枝杆菌临床分离株经PCR SSCP分析 ,4 1株RFP敏感株SSCP图谱与结核分枝杆菌标准株相同 ;70株耐RFP菌株中 ,6 3株(90 % )SSCP图谱与结核分枝杆菌标准株不同 ,其余 7株SSCP图谱与结核分枝杆菌标准株相同。基因阵列检测结果 4 1株RFP敏感株杂交图谱与标准株完全相同 ,70株耐RFP临床分离株中 ,6 3株检测到rpoB基因突变 ,检出率为 90 % ;其中 37株 (5 3% ) 5 31位丝氨酸 (Ser)置换 ,15株 (2 1% ) 5 2 6位组氨酸(His)置换 ,11株 (16 % )其他位置的氨基酸置换。基因阵列检测结果与PCR SSCP及测序结果一致。结论　用基因阵列法可简便、快速、准确地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变。     

Comparative assays of the rpoB gene for identification of Mycobacterium tuberculosis isolated from patients in Sudan.

OBJECTIVES: To characterise mycobacterial clinical isolates based on amplification of the rpoB gene. SETTING: One hundred and thirty-five mycobacterial isolates cultured from suspected pulmonary tuberculosis (TB) patients were identified phenotypically. Molecular characterisation of the isolates was performed based on amplification of the rpoB gene, using duplex polymerase chain reaction (DPCR), PCR-restriction fragment length polymorphism (RFLP) and nested PCR-based sequence analysis techniques. RESULTS: The DPCR assay identified 129 of 135 (95.5%) clinical isolates as Mycobacterium tuberculosis complex species. Restriction enzyme analysis of the rpoB PCR product using Hind II identified 134 of the 135 (99.3%) isolates as M. tuberculosis complex, while nested PCR sequence analysis of the rpoB gene identified 133/133 examined isolates (100%) as M. tuberculosis species. No mycobacteria other than M. tuberculosis (MOTT) were detected among the studied isolates. CONCLUSION: DPCR, PCR/RFLP Hind II and nested PCR sequence analysis of the rpoB gene techniques showed comparable efficiency in the characterisation of Mycobacterium isolates. Nested PCR sequence analysis of the rpoB gene was superior to PCR/RFLP for characterisation of suspected M. tuberculosis isolates, while the DPCR technique showed less sensitivity. As PCR-RFLP requires less sophisticated laboratory facilities than nested PCR sequence analysis, it would be more appropriate to be adopted for accurate characterisation of mycobacteria in countries with a weak infrastructure.     

结核分枝杆菌耐利福平药物机理的初步研究

目的通过诱导试验观察结核菌产生耐利福平药物的全过程,初步分析结核菌耐该药的机理。方法采用诱导试验、聚合酶链反应—单链构象多态性(PCR-SSCP)和序列分析,对经不同药物浓度传代培养后的结核菌强毒株（H₃₇Rv）和36株临床耐药分离株进行分析。结果在诱导到第7代时,H₃₇Rv的PCR-SSCP电泳出现异常,经测序证实在513位点发生基因突变。36株临床耐药分离株中有23株发生突变,为63.9% (23/36);其中有5株与诱导株基因突变位点相同。结论用药物不间断的刺激结核菌,是产生结核菌耐利福平药物的重要原因。     

Detecting genetic variability among different Mycobacterium tuberculosis strains using DNA microarrays technology

Recent advances in functional and comparative genomics have improved our understanding of genetic diversity among the Mycobacterium tuberculosis complex. In this study, we investigated the genetic polymorphism of M. tuberculosis using whole-genome microarray analysis. Amplified fragments of 15 M. tuberculosis strains (from two different geographical origins) and the reference strain H37Rv were produced by random amplification of polymorphic DNA (RAPD) using three different primers. The RAPD products were labeled with fluorescent dyes (Cy3 and Cy5) and hybridized to a TB DNA microarray representing nearly all open reading frames (ORFs) of H37Rv. The final results were analyzed using bioinformatic tools. Some genetic variability was found among the 16 M. tuberculosis strains. The majority of the highly polymorphic DNA sequences were observed in ORFs representing non-essential genes of the bacterium. The future use of comparative genomics based on DNA microarray technology should prove a powerful tool for understanding phenotypic variability among M. tuberculosis isolates of similar genetic composition. It is also a promising approach to provide important insights into evolution, virulence and pathogenesis of M. tuberculosis.